Antibodies directed against interleukin-33 (IL-33) and methods of making and using

ABSTRACT

The invention relates to an isolated immunoglobulin heavy chain polypeptide and an isolated immunoglobulin light chain polypeptide that bind to interleukin-33 (IL-33). The invention provides an IL-33-binding agent that comprises the aforementioned immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide. The invention also provides related vectors, compositions, and methods of using the IL-33-binding agent to treat a disorder in a mammal that is responsive to IL-33 inhibition.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 283,278 Byte ASCII (Text) file named“725934_ST25.txt,” created on Jul. 8, 2016.

BACKGROUND OF THE INVENTION

Interleukin 33 (IL-33), also known as nuclear factor (NF) in highendothelial venules (NF-HEV), is a cytokine belonging to the IL-1superfamily. IL-33 induces helper T cells, mast cells, eosinophils andbasophils to produce type 2 cytokines. IL-33 mediates its biologicaleffects by interacting with the receptors ST2 (also known as IL1RL1) andIL-1 Receptor Accessory Protein (IL1RAP) to activate intracellularmolecules in the NF-κB and MAP kinase signaling pathways that driveproduction of type 2 cytokines (e.g., IL-4, IL-5, and IL-13) frompolarized helper T cells (Th2) and Group-2 innate lymphoid cells (ILC2)in the skin, lungs, and gastrointestinal tract. IL-33 acts directly onmast cells to trigger their activation, and stimulates eosinophils andbasophils to degranulate, causing tissue damage. The induction of type 2cytokines by IL-33 in vivo is believed to induce the severe pathologicalchanges observed in mucosal organs following administration of IL-33(see, e.g., Schmitz et al., Immunity, 23(5): 479-490 (2005); andChackerian et al., J. Immunol., 179 (4): 2551-2555 (2007))

Both the in vivo expression profile of IL-33 and its cellular targetssuggest a role for IL-33 in Th2-driven pathologies. For example, IL-33expression has been detected in inflamed tissue from patients withmoderate-to-severe asthma, atopic dermatitis, allergic rhinitis, foodallergies, rheumatoid arthritis, multiple sclerosis, and Crohn'sdisease. In addition, functional single nucleotide polymorphisms (SNPs)in the distal promoter region of ST2 (IL-33R) have shown a significantassociation with atopic dermatitis (see, e.g., Shimizu et al., Hum. Mol.Genet., 14(19): 2919-2927 (2005)). Genome-wide association studies(GWAS) have also shown a strong link with SNPs in IL-33 and ST2 (IL-33R)genes for asthma in multiple studies of ethnically diverse groups (see,e.g., Gudbjartsson et al., Nat. Genet., 41(3): 342-347 (2009); Melén etal., J Allergy Clin. Immunol., 126(3): 631-637 (2010); Moffatt et al.,New Engl J. Med., 363(13):1211-1221 (2010); and Torgerson et al., Nat.Genet., 43(9): 887-92 (2011)). IL-33 (possibly in combination with IL-25and TSLP) also activates innate lymphoid cells (ILC2 cells) leading toTh2 cytokine secretion, anti-parasitic responses, and tissueimmunopathology.

Studies also suggest that IL-33 plays a direct role in some cancersexpressing the IL-33 receptor such as, for example, epithelial cancers(i.e., carcinomas) by acting as a survival or growth factor for cancercells. Such responsiveness to IL-33 might contribute to escape ofcertain cancer cell types from current standard of care (e.g., chronicmyelogenous leukemia (CML), breast cancers, and gastrointestinalcancers). Furthermore, IL-33 may play an indirect role in cancerprogression by reducing the protective activity of the immune system incontrolling tumor cells. Other recent studies suggest that IL-33 plays arole in the pathology of fibrosis, such as, for example, skin fibrosis,liver fibrosis, systemic sclerosis, and lung fibrosis. In addition,Mchedlidze et al., Immunity, 39: 357-371 (2013), demonstrates thathepatic expression of interleukin-33 (IL-33) is both required andsufficient for severe hepatic fibrosis in vivo.

Therefore, there is a need for inhibitors of IL-33 (e.g., antibodies)that bind IL-33 with high affinity and effectively neutralize IL-33activity. The invention provides an IL-33 binding agent that binds toand inhibits IL-33.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises (a) an amino acid sequence of any one of SEQID NO: 1, SEQ ID NO: 2, and SEQ ID NOs: 5-50, or (b) an amino acidsequence that is at least 90% identical to any one of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NOs: 5-50, SEQ ID NOs: 67-140, SEQ ID NO: 176, SEQ IDNO: 177, SEQ ID NOs: 178-188, and SEQ ID NOs: 206-217.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises (a) an amino acid sequence of any one of SEQID NO: 3, SEQ ID NO: 4, and SEQ ID NOs: 51-66, or (b) an amino acidsequence that is at least 90% identical to any one of SEQ ID NO: 3, SEQID NO: 4, SEQ ID NOs: 51-66, SEQ ID NOs: 141-175, SEQ ID NOs: 189-205,and SEQ ID NOs: 218-231.

In addition, the invention provides isolated or purified nucleic acidsequences encoding the foregoing immunoglobulin polypeptides, vectorscomprising such nucleic acid sequences, isolated IL-33-binding agentscomprising the foregoing immunoglobulin polypeptides, nucleic acidsequences encoding such IL-33-binding agents, vectors comprising suchnucleic acid sequences, isolated cells comprising such vectors,compositions comprising such IL-33-binding agents or such vectors with apharmaceutically acceptable carrier, and methods of treating a diseaseor disorder in mammals that is responsive to IL-33 inhibition orneutralization by administering effective amounts of such compositionsto mammals.

BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWINGS

FIG. 1A is a graph which depicts experimental data illustrating thedetermination of EC₅₀ for IL-33 stimulation of IL-5 secretion from KU812cells. FIG. 1B is a graph which depicts experimental data illustratingthat the inventive IL-33 binding agent inhibits IL-33-mediated releaseof IL-5 from KU812 cells.

FIG. 2A is a graph which depicts experimental data illustrating thatIL-33-induces expression of luciferase from the IL-8 promoter inHEK293-ST2 cells. FIG. 2B is a graph which depicts experimental dataillustrating that the inventive IL-33 binding agent inhibitsIL-33-induced expression of luciferase from the IL-8 promoter inHEK293-ST2 cells.

FIG. 3 is a graph which depicts experimental data illustrating that theinventive IL-33-binding agent inhibits IL-33-mediated release of IL-5 inprimary human basophils. For APE4909, IC50=2.2±1.1 nM (N=3); for ST2monomer, IC50=20 nM (N=1). The dashed lines marked “IL-33” and “medium”represent the concentration of IL-5 secreted in the absence of antibodyand in the absence of IL-33, respectively. The 0.02 suffix appended toAPE4909 refers to the lot of protein tested in these experiments.

FIG. 4 is a graph which depicts experimental data illustrating that theinventive IL-33-binding agent inhibits IL-33-mediated release of IL-9from primary human basophils. The IC₅₀ for APE4909 was measured at 3 nM.The dashed lines marked “IL-33” and “medium” represent the concentrationof IL-9 secreted in the absence of antibody and in the absence of IL-33,respectively. The antibody APE0422 represents an isotype controlantibody.

FIG. 5 is a graph which depicts experimental data illustrating affinityof the inventive APE4909 antibody for human IL-33 as measured byKINEXA™. Results indicate KD=1.0 pM (N=2) with 95% confidence interval(CI) of 1.9 pM-420 fM.

FIG. 6 is a graph which depicts experimental data illustrating affinityof the inventive APE4909 antibody for cynomolgus IL-33 as measured byKINEXA™.

FIG. 7 is a graph which depicts experimental data illustrating theability of the inventive APE4909 antibody to inhibit human IL-33-driveneosinophil expansion in the peripheral blood compartment.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated immunoglobulin heavy chainpolypeptide and/or an isolated immunoglobulin light chain polypeptide,or a fragment (e.g., antigen-binding fragment) thereof. The term“immunoglobulin” or “antibody,” as used herein, refers to a protein thatis found in blood or other bodily fluids of vertebrates, which is usedby the immune system to identify and neutralize foreign objects, such asbacteria and viruses. The polypeptide is “isolated” in that it isremoved from its natural environment. In a preferred embodiment, animmunoglobulin or antibody is a protein that comprises at least onecomplementarity determining region (CDR). The CDRs form the“hypervariable region” of an antibody, which is responsible for antigenbinding (discussed further below). A whole immunoglobulin typicallyconsists of four polypeptides: two identical copies of a heavy (H) chainpolypeptide and two identical copies of a light (L) chain polypeptide.Each of the heavy chains contains one N-terminal variable (V_(H)) regionand three C-terminal constant (C_(H)1, C_(H)2, and C_(H)3) regions, andeach light chain contains one N-terminal variable (V_(L)) region and oneC-terminal constant (CO region. The light chains of antibodies can beassigned to one of two distinct types, either kappa (κ) or lambda (λ),based upon the amino acid sequences of their constant domains. In atypical immunoglobulin, each light chain is linked to a heavy chain bydisulphide bonds, and the two heavy chains are linked to each other bydisulphide bonds. The light chain variable region is aligned with thevariable region of the heavy chain, and the light chain constant regionis aligned with the first constant region of the heavy chain. Theremaining constant regions of the heavy chains are aligned with eachother.

The variable regions of each pair of light and heavy chains form theantigen binding site of an antibody. The V_(H) and V_(L) regions havethe same general structure, with each region comprising four framework(FW or FR) regions. The term “framework region,” as used herein, refersto the relatively conserved amino acid sequences within the variableregion which are located between the hypervariable or complementarydetermining regions (CDRs). There are four framework regions in eachvariable domain, which are designated FR1, FR2, FR3, and FR4. Theframework regions form the β sheets that provide the structuralframework of the variable region (see, e.g., C. A. Janeway et al.(eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y.(2001)).

The framework regions are connected by three complementarity determiningregions (CDRs). As discussed above, the three CDRs, known as CDR1, CDR2,and CDR3, form the “hypervariable region” of an antibody, which isresponsible for antigen binding. The CDRs form loops connecting, and insome cases comprising part of, the beta-sheet structure formed by theframework regions. While the constant regions of the light and heavychains are not directly involved in binding of the antibody to anantigen, the constant regions can influence the orientation of thevariable regions. The constant regions also exhibit various effectorfunctions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells.

The isolated immunoglobulin heavy chain polypeptide and the isolatedimmunoglobulin light chain polypeptide of the invention desirably bindto IL-33. As discussed above, interleukin-33 (IL-33) (also known asnuclear factor (NF) in high endothelial venules (NF-HEV)) is a cytokineof the IL-1 family, which also includes the inflammatory cytokinesIL-1α, IL-1β, and IL-18. IL-33 has been shown to signal via the ST2receptor and the IL1RAP receptor. IL-33 is expressed broadly in varioustissues, including stomach, lung, spinal cord, brain, and skin, as wellas in cells, including smooth muscle cells and epithelial cells liningbronchus and small airways. IL-33 expression is induced by IL-1β andtumor necrosis factor-α (TNF-α) in lung and dermal fibroblasts and, to alesser extent, by macrophage activation. IL-33 treatment has been shownto induce T-helper (Th) type 2 responses in mice as indicated by anincrease in Th2 cytokine production and serum immunoglobulin. Systemictreatment of mice with IL-33 results in pathologic changes in the lungand the digestive tract (see, e.g., Choi et al., Blood, 114(14):3117-3126 (2009); and Yagami et al., J. Immunology, 185(10): 5743-5750(2010)).

IL-33 is produced as a 30-kDa precursor protein that is cleaved in vitroby caspase-1, releasing the mature 18-kDa form (see, e.g., Schmitz etal., Immunity, 23(5): 479-490 (2005)). Upon binding to the ST2 receptor,IL-33 promotes the activation of nuclear factor (NF)-κB andmitogen-activated protein kinase (MAPK), leading to increasedtranscription of Th2 cytokines (Schmitz et al., supra).

Antibodies which bind to IL-33, and components thereof, are known in theart (see, e.g., U.S. Patent Application Publications 2009/0041718 A1 and2012/0263709 A1). Anti-IL-33 antibodies also are commercially availablefrom sources such as, for example, Abcam (Cambridge, Mass.).

The invention provides an immunoglobulin heavy chain polypeptide thatcomprises an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NOs: 5-50, SEQ ID NOs: 67-140, SEQ ID NO: 176, SEQ ID NO: 177,SEQ ID NOs: 178-188, and SEQ ID NOs: 206-217, or an amino acid sequencethat is at least 90% identical to any one of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NOs: 5-50, SEQ ID NOs: 67-140, SEQ ID NO: 176, SEQ ID NO: 177,SEQ ID NOs: 178-188, and SEQ ID NOs: 206-217. In one embodiment of theinvention, the isolated immunoglobulin heavy chain polypeptidecomprises, consists of, or consists essentially of an amino acidsequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NOs: 5-50, SEQID NOs: 67-140, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NOs: 178-188, andSEQ ID NOs: 206-217. When the inventive immunoglobulin heavy chainpolypeptide consists essentially of an amino acid sequence of any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NOs: 5-50, SEQ ID NOs: 67-140, SEQ IDNO: 176, SEQ ID NO: 177, SEQ ID NOs: 178-188, and SEQ ID NOs: 206-217,additional components can be included in the polypeptide that do notmaterially affect the polypeptide (e.g., protein moieties such as biotinthat facilitate purification or isolation). When the inventiveimmunoglobulin heavy chain polypeptide consists of an amino acidsequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NOs: 5-50, SEQID NOs: 67-140, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NOs: 178-188, andSEQ ID NOs: 206-217, the polypeptide does not comprise any additionalcomponents (i.e., components that are not endogenous to the inventiveimmunoglobulin heavy chain polypeptide).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NOs:5-50, SEQ ID NOs: 67-140, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NOs:178-188, or SEQ ID NOs: 206-217. Nucleic acid or amino acid sequence“identity,” as described herein, can be determined by comparing anucleic acid or amino acid sequence of interest to a reference nucleicacid or amino acid sequence. The percent identity is the number ofnucleotides or amino acid residues that are the same (i.e., that areidentical) as between the sequence of interest and the referencesequence divided by the length of the longest sequence (i.e., the lengthof either the sequence of interest or the reference sequence, whicheveris longer). A number of mathematical algorithms for obtaining theoptimal alignment and calculating identity between two or more sequencesare known and incorporated into a number of available software programs.Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (foralignment of nucleic acid and amino acid sequences), BLAST programs(e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs(e.g., FASTA3×, FAS™, and SSEARCH) (for sequence alignment and sequencesimilarity searches). Sequence alignment algorithms also are disclosedin, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410(1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775(2009), Durbin et al., eds., Biological Sequence Analysis: ProbabilisticModels of Proteins and Nucleic Acids, Cambridge University Press,Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005),Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), andGusfield, Algorithms on Strings, Trees and Sequences, CambridgeUniversity Press, Cambridge UK (1997)).

The invention provides an immunoglobulin light chain polypeptide thatcomprises an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NOs: 51-66, SEQ ID NOs: 141-175, SEQ ID NOs: 189-205, and SEQID NOs: 218-231, or an amino acid sequence that is at least 90%identical to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs: 51-66,SEQ ID NOs: 141-175, SEQ ID NOs: 189-205, and SEQ ID NOs: 218-231. Inone embodiment of the invention, the isolated immunoglobulin light chainpolypeptide comprises, consists of, or consists essentially of an aminoacid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs:51-66, SEQ ID NOs: 141-175, SEQ ID NOs: 189-205, and SEQ ID NOs:218-231. When the inventive immunoglobulin light chain polypeptideconsists essentially of an amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NOs: 51-66, SEQ ID NOs: 141-175, SEQ ID NOs:189-205, and SEQ ID NOs: 218-231, additional components can be includedin the polypeptide that do not materially affect the polypeptide (e.g.,protein moieties such as biotin that facilitate purification orisolation). When the inventive immunoglobulin light chain polypeptideconsists of an amino acid sequence of any one of SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NOs: 51-66, SEQ ID NOs: 141-175, SEQ ID NOs: 189-205, andSEQ ID NOs: 218-231, the polypeptide does not comprise any additionalcomponents (i.e., components that are not endogenous to the inventiveimmunoglobulin light chain polypeptide).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NOs:51-66, SEQ ID NOs: 141-175, SEQ ID NOs: 189-205, or SEQ ID NOs: 218-231.Nucleic acid or amino acid sequence “identity,” as described herein, canbe determined using the methods described herein.

One or more amino acids of the aforementioned immunoglobulin heavy chainpolypeptides and/or light chain polypeptides can be replaced orsubstituted with a different amino acid. An amino acid “replacement” or“substitution” refers to the replacement of one amino acid at a givenposition or residue by another amino acid at the same position orresidue within a polypeptide sequence.

Amino acids are broadly grouped as “aromatic” or “aliphatic.” Anaromatic amino acid includes an aromatic ring. Examples of “aromatic”amino acids include histidine (H or His), phenylalanine (F or Phe),tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acidsare broadly grouped as “aliphatic.” Examples of “aliphatic” amino acidsinclude glycine (G or Gly), alanine (A or Ala), valine (V or Val),leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine(S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P orPro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (Nor Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R orArg).

Aliphatic amino acids may be sub-divided into four sub-groups. The“large aliphatic non-polar sub-group” consists of valine, leucine, andisoleucine. The “aliphatic slightly-polar sub-group” consists ofmethionine, serine, threonine, and cysteine. The “aliphaticpolar/charged sub-group” consists of glutamic acid, aspartic acid,asparagine, glutamine, lysine, and arginine. The “small-residuesub-group” consists of glycine and alanine. The group of charged/polaramino acids may be sub-divided into three sub-groups: the“positively-charged sub-group” consisting of lysine and arginine, the“negatively-charged sub-group” consisting of glutamic acid and asparticacid, and the “polar sub-group” consisting of asparagine and glutamine.

Aromatic amino acids may be sub-divided into two sub-groups: the“nitrogen ring sub-group” consisting of histidine and tryptophan and the“phenyl sub-group” consisting of phenylalanine and tyrosine.

The amino acid replacement or substitution can be conservative,semi-conservative, or non-conservative. The phrase “conservative aminoacid substitution” or “conservative mutation” refers to the replacementof one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz andSchirmer, Principles of Protein Structure, Springer-Verlag, New York(1979)). According to such analyses, groups of amino acids may bedefined where amino acids within a group exchange preferentially witheach other, and therefore resemble each other most in their impact onthe overall protein structure (Schulz and Schirmer, supra).

Examples of conservative amino acid substitutions include substitutionsof amino acids within the sub-groups described above, for example,lysine for arginine and vice versa such that a positive charge may bemaintained, glutamic acid for aspartic acid and vice versa such that anegative charge may be maintained, serine for threonine such that a free—OH can be maintained, and glutamine for asparagine such that a free—NH₂ can be maintained.

“Semi-conservative mutations” include amino acid substitutions of aminoacids within the same groups listed above, but not within the samesub-group. For example, the substitution of aspartic acid forasparagine, or asparagine for lysine, involves amino acids within thesame group, but different sub-groups. “Non-conservative mutations”involve amino acid substitutions between different groups, for example,lysine for tryptophan, or phenylalanine for serine, etc.

In addition, one or more amino acids can be inserted into theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides. Any number of any suitable amino acids can beinserted into the amino acid sequence of the immunoglobulin heavy chainpolypeptide and/or light chain polypeptide. In this respect, at leastone amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids),but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 orless amino acids), can be inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids) are inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.In this respect, the amino acid(s) can be inserted into any one of theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides in any suitable location. Preferably, the aminoacid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.

The inventive isolated immunoglobulin heavy chain polypeptide and lightchain polypeptides are not limited to polypeptides comprising thespecific amino acid sequences described herein. Indeed, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that competeswith the inventive immunoglobulin heavy chain polypeptide or light chainpolypeptide for binding to IL-33. In this respect, for example, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that binds to thesame epitope of IL-33 recognized by the heavy and light chainpolypeptides described herein. Antibody competition can be assayed usingroutine peptide competition assays which utilize ELISA, Western blot, orimmunohistochemistry methods (see, e.g., U.S. Pat. Nos. 4,828,981 and8,568,992; and Braitbard et al., Proteome Sci., 4: 12 (2006)).

The invention provides an isolated interleukin-33 (IL-33)-binding agentcomprising, consisting essentially of, or consisting of one or more ofthe inventive isolated amino acid sequences described herein. By“interleukin-33 (IL-33)-binding agent” is meant a molecule, preferably aproteinaceous molecule, that binds specifically to IL-33. Preferably,the IL-33-binding agent is an antibody or a fragment (e.g., immunogenicfragment) thereof. The isolated IL-33-binding agent of the inventioncomprises, consists essentially of, or consists of the inventiveisolated immunoglobulin heavy chain polypeptide and/or the inventiveisolated immunoglobulin light chain polypeptide. In one embodiment, theisolated IL-33-binding agent comprises, consists essentially of, orconsists of the inventive immunoglobulin heavy chain polypeptide or theinventive immunoglobulin light chain polypeptide. In another embodiment,the isolated IL-33-binding agent comprises, consists essentially of, orconsists of the inventive immunoglobulin heavy chain polypeptide and theinventive immunoglobulin light chain polypeptide.

Any amino acid residue of the inventive immunoglobulin heavy chainpolypeptide and/or the inventive immunoglobulin light chain polypeptidecan be replaced, in any combination, with a different amino acidresidue, or can be deleted or inserted, so long as the biologicalactivity of the IL-33-binding agent is enhanced or improved as a resultof the amino acid replacements, insertions, and/or deletions. The“biological activity” of an IL-33-binding agent refers to, for example,binding affinity for a particular IL-33 epitope, neutralization orinhibition of IL-33 binding to its receptor(s), neutralization orinhibition of IL-33 activity in vivo (e.g., IC₅₀), pharmacokinetics, andcross-reactivity (e.g., with non-human homologs or orthologs of theIL-33 protein, or with other proteins or tissues). Other biologicalproperties or characteristics of an antigen-binding agent recognized inthe art include, for example, avidity, selectivity, solubility, folding,immunotoxicity, expression, and formulation. The aforementionedproperties or characteristics can be observed, measured, and/or assessedusing standard techniques including, but not limited to, ELISA,competitive ELISA, surface plasmon resonance analysis (BIACORE™), orKINEXA™, in vitro or in vivo neutralization assays, receptor-ligandbinding assays, cytokine or growth factor production and/or secretionassays, and signal transduction and immunohistochemistry assays.

The terms “inhibit” or “neutralize,” as used herein with respect to theactivity of a IL-33-binding agent, refer to the ability to substantiallyantagonize, prohibit, prevent, restrain, slow, disrupt, alter,eliminate, stop, or reverse the progression or severity of, for example,the biological activity of IL-33, or a disease or condition associatedwith IL-33. The isolated IL-33-binding agent of the invention preferablyinhibits or neutralizes the activity of IL-33 by at least about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 100%, or a range defined by any two of theforegoing values.

The isolated IL-33-binding agent of the invention can be a wholeantibody, as described herein, or an antibody fragment. The Willis“fragment of an antibody,” “antibody fragment,” and “functional fragmentof an antibody” are used interchangeably herein to mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen (see, generally, Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). The isolated IL-33 binding agent can contain anyIL-33-binding antibody fragment. The antibody fragment desirablycomprises, for example, one or more CDRs, the variable region (orportions thereof), the constant region (or portions thereof), orcombinations thereof. Examples of antibody fragments include, but arenot limited to, (i) a Fab fragment, which is a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and CH₁ domains, (ii) a F(ab′)₂fragment, which is a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region, (iii) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (iv) a Fab′ fragment, which results from breaking thedisulfide bridge of an F(ab′)₂ fragment using mild reducing conditions,(v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domainantibody (dAb), which is an antibody single variable region domain (VHor VL) polypeptide that specifically binds antigen.

In embodiments where the isolated IL-33-binding agent comprises afragment of the immunoglobulin heavy chain or light chain polypeptide,the fragment can be of any size so long as the fragment binds to, andpreferably inhibits the activity of, IL-33. In this respect, a fragmentof the immunoglobulin heavy chain polypeptide desirably comprisesbetween about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, or a range defined by any two of the foregoing values)amino acids. Similarly, a fragment of the immunoglobulin light chainpolypeptide desirably comprises between about 5 and 18 (e.g., about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined byany two of the foregoing values) amino acids.

When the IL-33-binding agent is an antibody or antibody fragment, theantibody or antibody fragment desirably comprises a heavy chain constantregion (F_(c)) of any suitable class. Preferably, the antibody orantibody fragment comprises a heavy chain constant region that is basedupon wild-type IgG1, IgG2, or IgG4 antibodies, or variants thereof.

The IL-33-binding agent also can be a single chain antibody fragment.Examples of single chain antibody fragments include, but are not limitedto, (i) a single chain Fv (scFv), which is a monovalent moleculeconsisting of the two domains of the Fv fragment (i.e., V_(L) and V_(H))joined by a synthetic linker which enables the two domains to besynthesized as a single polypeptide chain (see, e.g., Bird et al.,Science, 242: 423-426 (1988); Huston et al., Proc. Mid Acad. Sci. USA,85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778(1998)) and (ii) a diabody, which is a dimer of polypeptide chains,wherein each polypeptide chain comprises a V_(H) connected to a V_(L) bya peptide linker that is too short to allow pairing between the V_(H)and V_(L) on the same polypeptide chain, thereby driving the pairingbetween the complementary domains on different V_(H)-V_(L) polypeptidechains to generate a dimeric molecule having two functional antigenbinding sites. Antibody fragments are known in the art and are describedin more detail in, e.g., U.S. Patent Application Publication2009/0093024 A1.

The isolated IL-33-binding agent also can be an intrabody or fragmentthereof. An intrabody is an antibody which is expressed and whichfunctions intracellularly. Intrabodies typically lack disulfide bondsand are capable of modulating the expression or activity of target genesthrough their specific binding activity. Intrabodies include singledomain fragments such as isolated V_(H) and V_(L) domains and scFvs. Anintrabody can include sub-cellular trafficking signals attached to the Nor C terminus of the intrabody to allow expression at highconcentrations in the sub-cellular compartments where a target proteinis located. Upon interaction with a target gene, an intrabody modulatestarget protein function and/or achieves phenotypic/functional knockoutby mechanisms such as accelerating target protein degradation andsequestering the target protein in a non-physiological sub-cellularcompartment. Other mechanisms of intrabody-mediated gene inactivationcan depend on the epitope to which the intrabody is directed, such asbinding to the catalytic site on a target protein or to epitopes thatare involved in protein-protein, protein-DNA, or protein-RNAinteractions.

The isolated IL-33-binding agent also can be an antibody conjugate. Inthis respect, the isolated IL-33-binding agent can be a conjugate of (1)an antibody, an alternative scaffold, or fragments thereof, and (2) aprotein or non-protein moiety comprising the IL-33-binding agent. Forexample, the IL-33-binding agent can be all or part of an antibodyconjugated to a peptide, a fluorescent molecule, or a chemotherapeuticagent.

The isolated IL-33-binding agent can be, or can be obtained from, ahuman antibody, a non-human antibody, or a chimeric antibody. By“chimeric” is meant an antibody or fragment thereof comprising bothhuman and non-human regions. Preferably, the isolated IL-33-bindingagent is a humanized antibody. A “humanized” antibody is a monoclonalantibody comprising a human antibody scaffold and at least one CDRobtained or derived from a non-human antibody. Non-human antibodiesinclude antibodies isolated from any non-human animal, such as, forexample, a rodent (e.g., a mouse or rat). A humanized antibody cancomprise, one, two, or three CDRs obtained or derived from a non-humanantibody. In one embodiment of the invention, CDRH3 of the inventiveIL-33-binding agent is obtained or derived from a mouse monoclonalantibody, while the remaining variable regions and constant region ofthe inventive IL-33-binding agent are obtained or derived from a humanmonoclonal antibody.

A human antibody, a non-human antibody, a chimeric antibody, or ahumanized antibody can be obtained by any means, including via in vitrosources (e.g., a hybridoma or a cell line producing an antibodyrecombinantly) and in vivo sources (e.g., rodents). Methods forgenerating antibodies are known in the art and are described in, forexample, Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976);Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press(1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., GarlandPublishing, New York, N.Y. (2001)). In certain embodiments, a humanantibody or a chimeric antibody can be generated using a transgenicanimal (e.g., a mouse) wherein one or more endogenous immunoglobulingenes are replaced with one or more human immunoglobulin genes. Examplesof transgenic mice wherein endogenous antibody genes are effectivelyreplaced with human antibody genes include, but are not limited to, theMedarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™(see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), andLonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanizedantibody can be generated using any suitable method known in the art(see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Benchto Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including,e.g., grafting of non-human CDRs onto a human antibody scaffold (see,e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J.Biochem., 144(1): 115-120 (2008)). In one embodiment, a humanizedantibody can be produced using the methods described in, e.g., U.S.Patent Application Publication 2011/0287485 A1.

In one embodiment, a CDR (e.g., CDR1, CDR2, or CDR3) or a variableregion of the immunoglobulin heavy chain polypeptide and/or theimmunoglobulin light chain polypeptide described herein can betransplanted (i.e., grafted) into another molecule, such as an antibodyor non-antibody polypeptide, using either protein chemistry orrecombinant DNA technology. In this regard, the invention provides anisolated IL-33-binding agent comprising at least one CDR of animmunoglobulin heavy chain and/or light chain polypeptide as describedherein. The isolated IL-33-binding agent can comprise one, two, or threeCDRs of an immunoglobulin heavy chain and/or light chain variable regionas described herein. For example, with respect to immunoglobulin heavychain polypeptides comprising any one of SEQ ID NO: 1, SEQ ID NO: 2, orSEQ ID NOs: 5-50, the CDR1 is located between amino acid residues 26 and35, inclusive; the CDR2 is located between amino acid residues 50 and59, inclusive (SEQ ID NO: 1 and SEQ ID NO: 2) or between amino acidresidues 50 and 66, inclusive (SEQ ID NOs: 5-50); and the CDR3 islocated between amino acid residues 99 and 102, inclusive (SEQ ID NO: 1and SEQ ID NO: 2) or between amino acid residues 99 and 111, inclusive(SEQ ID NOs 5-50). With respect to immunoglobulin light chainpolypeptides comprising any one of SEQ ID NO: 3, SEQ ID NO: 4, and SEQID NO: 51-66, for example, the CDR1 is located between amino acidresidues 24 and 39, inclusive (SEQ ID NO: 3 and SEQ ID NO: 4) or betweenamino acid residues 24 and 34, inclusive (SEQ ID NOs: 51-66); the CDR2is located between amino acid residues 55 and 61, inclusive (SEQ ID NO:3 and SEQ ID NO: 4) or between amino acid residues 50 and 56, inclusive(SEQ ID NOs: 51-66); the CDR3 is located between amino acid residues 94and 102, inclusive (SEQ ID NO: 3 and SEQ ID NO: 4) or between amino acidresidues 89 and 97, inclusive (SEQ ID NOs: 51-66).

In a preferred embodiment, the IL-33-binding agent binds an epitope ofIL-33 which blocks the binding of IL-33 to receptors ST2 (also known asIL1RL1) and/or IL-1 Receptor Accessory Protein (IL1RAP) and inhibitsIL-33 mediated signaling. The invention also provides an isolated orpurified epitope of IL-33 which blocks the binding of IL-33 to receptorsST2 and IL1RAP in an indirect or allosteric manner.

The invention also provides one or more isolated or purified nucleicacid sequences that encode the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide, andthe inventive IL-33-binding agent.

The term “nucleic acid sequence” is intended to encompass a polymer ofDNA or RNA, i.e., a polynucleotide, which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “nucleic acid” and “polynucleotide” as usedherein refer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated and/or capped polynucleotides. Nucleic acids aretypically linked via phosphate bonds to form nucleic acid sequences orpolynucleotides, though many other linkages are known in the art (e.g.,phosphorothioates, boranophosphates, and the like).

The invention further provides a vector comprising one or more nucleicacid sequences encoding the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide,and/or the inventive IL-33-binding agent. The vector can be, forexample, a plasmid, episome, cosmid, viral vector (e.g., retroviral oradenoviral), or phage. Suitable vectors and methods of vectorpreparation are well known in the art (see, e.g., Sambrook et al.,Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994)).

In addition to the nucleic acid sequence encoding the inventiveimmunoglobulin heavy polypeptide, the inventive immunoglobulin lightchain polypeptide, and/or the inventive IL-33-binding agent, the vectorpreferably comprises expression control sequences, such as promoters,enhancers, polyadenylation signals, transcription terminators, internalribosome entry sites (IRES), and the like, that provide for theexpression of the coding sequence in a host cell. Exemplary expressioncontrol sequences are known in the art and described in, for example,Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185,Academic Press, San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, the RSV promoter. Inducible promoters include, forexample, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), theEcdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.),LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERTtamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res.,27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No.7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308: 123-144(2005)).

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences.

The vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allow cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/008796 and WO 1994/028143; Wigler et al., Proc. Natl. Acad. Sci.USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA,78: 1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1-14(1981); Santerre et al., Gene, 30: 147-156 (1984); Kent et al., Science,237: 901-903 (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026-2034 (1962); Lowy etal., Cell, 22: 817-823 (1980); and U.S. Pat. Nos. 5,122,464 and5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al., Gene Therapy,11: 1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

Other suitable vectors include integrating expression vectors, which mayrandomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system fromInvitrogen (Carlsbad, Calif.) (e.g., pcDNA™5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Life Technologies(Carlsbad, Calif.), UCOE from Millipore (Billerica, Mass.), and pCI orpFN10A (ACT) FLEXI™ from Promega (Madison, Wis.).

Viral vectors also can be used. Representative commercially availableviral expression vectors include, but are not limited to, theadenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, TheNetherlands), the lentiviral-based pLP 1 from Invitrogen (Carlsbad,Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH fromStratagene (La Jolla, Calif.).

Nucleic acid sequences encoding the inventive amino acid sequences canbe provided to a cell on the same vector (i.e., in cis). Aunidirectional promoter can be used to control expression of eachnucleic acid sequence. In another embodiment, a combination ofbidirectional and unidirectional promoters can be used to controlexpression of multiple nucleic acid sequences. Nucleic acid sequencesencoding the inventive amino acid sequences alternatively can beprovided to the population of cells on separate vectors (i.e., intrans). Each of the nucleic acid sequences in each of the separatevectors can comprise the same or different expression control sequences.The separate vectors can be provided to cells simultaneously orsequentially.

The vector(s) comprising the nucleic acid(s) encoding the inventiveamino acid sequences can be introduced into a host cell that is capableof expressing the polypeptides encoded thereby, including any suitableprokaryotic or eukaryotic cell. As such, the invention provides anisolated cell comprising the inventive vector. Preferred host cells arethose that can be easily and reliably grown, have reasonably fast growthrates, have well characterized expression systems, and can betransfoimed or transfected easily and efficiently.

Examples of suitable prokaryotic cells include, but are not limited to,cells from the genera Bacillus (such as Bacillus subtilis and Bacillusbrevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces,Salmonella, and Erwinia. Particularly useful prokaryotic cells includethe various strains of Escherichia coli (e.g., K12, HB101 (ATCC No.33694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

Preferably, the vector is introduced into a eukaryotic cell. Suitableeukaryotic cells are known in the art and include, for example, yeastcells, insect cells, and mammalian cells. Examples of suitable yeastcells include those from the genera Kluyveromyces, Pichia,Rhinosporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeastcells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Suitable insect cells are described in, for example, Kitts et al.,Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993).Preferred insect cells include Sf-9 and HIS (Invitrogen, Carlsbad,Calif.).

Preferably, mammalian cells are utilized in the invention. A number ofsuitable mammalian host cells are known in the art, and many areavailable from the American Type Culture Collection (ATCC, Manassas,Va.). Examples of suitable mammalian cells include, but are not limitedto, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells(Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), humanembryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3cells (ATCC No. CCL92). Other suitable mammalian cell lines are themonkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651),as well as the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Other suitable mammalian cell linesinclude, but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, and BHK or HaK hamster cell lines, all of which areavailable from the ATCC. Methods for selecting suitable mammalian hostcells and methods for transformation, culture, amplification, screening,and purification of cells are known in the art.

Most preferably, the mammalian cell is a human cell. For example, themammalian cell can be a human lymphoid or lymphoid derived cell line,such as a cell line of pre-B lymphocyte origin. Examples of humanlymphoid cells lines include, without limitation, RAMOS (CRL-1596),Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al.,Proc. Natl. Acad. Sci. USA, 85: 1581-1585 (1988)), Raji cells (CCL-86),and derivatives thereof.

A nucleic acid sequence encoding the inventive amino acid sequence maybe introduced into a cell by “transfection,” “transformation,” or“transduction.” “Transfection,” “transformation,” or “transduction,” asused herein, refer to the introduction of one or more exogenouspolynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art and include, forexample, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J.(ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression Protocols, Humana Press (1991)); DEAE-dextran;electroporation; cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors canbe introduced into host cells, after growth of infectious particles insuitable packaging cells, many of which are commercially available.

The invention provides a composition comprising an effective amount ofthe inventive immunoglobulin heavy chain polypeptide, the inventiveimmunoglobulin light chain polypeptide, the inventive IL-33-bindingagent, the inventive nucleic acid sequence encoding any of theforegoing, or the inventive vector comprising the inventive nucleic acidsequence. Preferably, the composition is a pharmaceutically acceptable(e.g., physiologically acceptable) composition, which comprises acarrier, preferably a pharmaceutically acceptable (e.g., physiologicallyacceptable) carrier, and the inventive amino acid sequences,antigen-binding agent, or vector. Any suitable carrier can be usedwithin the context of the invention, and such carriers are well known inthe art. The choice of carrier will be determined, in part, by theparticular site to which the composition may be administered and theparticular method used to administer the composition. The compositionoptionally can be sterile. The composition can be frozen or lyophilizedfor storage and reconstituted in a suitable sterile carrier prior touse. The compositions can be generated in accordance with conventionaltechniques described in, e.g., Remington: The Science and Practice ofPharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa.(2001).

The invention further provides a method of treating a disease ordisorder in a mammal that is responsive to IL-33 inhibition orneutralization. The method comprises administering the aforementionedcomposition to a mammal having a disease or disorder that is responsiveto IL-33 inhibition or neutralization, whereupon the disease disorder istreated in the mammal. A disease or disorder that is “responsive toIL-33 inhibition” or “responsive to IL-33 neutralization,” refers to anydisease or disorder in which a decrease in IL-33 levels or activity hasa therapeutic benefit in mammals, preferably humans, or the improperexpression (e.g., overexpression) or increased activity of IL-33 causesor contributes to the pathological effects of the disease or disorder.Diseases or disorders that are responsive to IL-33 inhibition orneutralization include, for example, inflammatory disorders, autoimmunediseases, certain cancers (e.g., epithelial cancers (carcinomas),chronic myelogenous leukemia (CML), breast cancers, and gastrointestinalcancers), and any atopic disorder. Inflammatory disorders include, forexample, allergic inflammation of the skin, lungs, and gastrointestinaltract, atopic dermatitis (also known as atopic eczema), asthma (allergicand non-allergic), fibrosis (e.g., idiopathic pulmonary fibrosis,scleroderma, kidney fibrosis, and scarring), chronic obstructivepulmonary disease (COPD), allergic rhinitis, food allergies (e.g.,allergies to peanuts, eggs, dairy, shellfish, tree nuts, etc.), seasonalallergies, and other allergies. Autoimmune diseases include, forexample, Crohn's disease, rheumatoid arthritis, psoriasis, ankylosingspondylitis, lupus erythematosus, and scleroderma. The term “atopic,” asused herein, refers to a hereditary predisposition toward developingcertain hypersensitivity reactions (e.g., eczema (atopic dermatitis),hay fever (allergic rhinitis), and allergy-induced asthma (allergicasthma)), which is typically mediated by excessive IgE production.

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. Preferably,the effect is therapeutic, i.e., the effect partially or completelycures a disease and/or adverse symptom attributable to the disease. Tothis end, the inventive method comprises administering a“therapeutically effective amount” of the IL-33-binding agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the IL-33-binding agent to elicit adesired response in the individual. For example, a therapeuticallyeffective amount of an IL-33-binding agent of the invention is an amountwhich decreases IL-33 bioactivity in a human.

Alternatively, the pharmacologic and/or physiologic effect may beprophylactic, i.e., the effect completely or partially prevents adisease or symptom thereof. In this respect, the inventive methodcomprises administering a “prophylactically effective amount” of theIL-33-binding agent. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve a desired prophylactic result (e.g., prevention of diseaseonset).

A typical dose can be, for example, in the range of 1 pg/kg to 20 mg/kgof animal or human body weight; however, doses below or above thisexemplary range are within the scope of the invention. The dailyparenteral dose can be about 0.00001 μg/kg to about 20 mg/kg of totalbody weight (e.g., about 0.001 μg/kg, about 0.1 μg/kg, about 1 μg/kg,about 5 μg/kg, about 10 μg/kg, about 100 μg/kg, about 500 μg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two ofthe foregoing values), preferably from about 0.1 μg/kg to about 10 mg/kgof total body weight (e.g., about 0.5 μg/kg, about 1 μg/kg, about 50μg/kg, about 150 μg/kg, about 300 μg/kg, about 750 μg/kg, about 1.5mg/kg, about 5 mg/kg, or a range defined by any two of the foregoingvalues), more preferably from about 1 μg/kg to 5 mg/kg of total bodyweight (e.g., about 3 μg/kg, about 15 μg/kg, about 75 μg/kg, about 300μg/kg, about 900 μg/kg, about 2 mg/kg, about 4 mg/kg, or a range definedby any two of the foregoing values), and even more preferably from about0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about 2.5mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg,about 13 mg/kg, or a range defined by any two of the foregoing values).Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and are within the scope ofthe invention. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The composition comprising an effective amount of the inventiveimmunoglobulin heavy chain polypeptide, the inventive immunoglobulinlight chain polypeptide, the inventive IL-33-binding agent, theinventive nucleic acid sequence encoding any of the foregoing, or theinventive vector comprising the inventive nucleic acid sequence can beadministered to a mammal using standard administration techniques,including oral, intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. The composition preferably is suitable forparenteral administration. The term “parenteral,” as used herein,includes intravenous, intramuscular, subcutaneous, rectal, vaginal, andintraperitoneal administration. More preferably, the composition isadministered to a mammal using peripheral systemic delivery byintravenous, intraperitoneal, or subcutaneous injection.

Once administered to a mammal (e.g., a cross-reactive human), thebiological activity of the inventive IL-33-binding agent can be measuredby any suitable method known in the art. For example, the biologicalactivity can be assessed by determining the stability of a particularIL-33-binding agent. In one embodiment of the invention, theIL-33-binding agent (e.g., an antibody) has an in vivo half life betweenabout 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes,about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10hours, about 12 hours, about 1 day, about 5 days, about 10 days, about15 days, about 25 days, about 35 days, about 40 days, about 45 days, ora range defined by any two of the foregoing values). In anotherembodiment, the IL-33-binding agent has an in vivo half life betweenabout 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15hours, about 20 hours, about 2 days, about 3 days, about 7 days, about12 days, about 14 days, about 17 days, about 19 days, or a range definedby any two of the foregoing values). In another embodiment, theIL-33-binding agent has an in vivo half life between about 10 days andabout 40 days (e.g., about 10 days, about 13 days, about 16 days, about18 days, about 20 days, about 23 days, about 26 days, about 29 days,about 30 days, about 33 days, about 37 days, about 38 days, about 39days, about 40 days, or a range defined by any two of the foregoingvalues).

The biological activity of a particular IL-33-binding agent also can beassessed by determining its binding affinity to IL-33 or an epitopethereof. The term “affinity” refers to the equilibrium constant for thereversible binding of two agents and is expressed as the dissociationconstant (K_(D)). Affinity of a binding agent to a ligand, such asaffinity of an antibody for an epitope, can be, for example, from about1 femtomolar (fM) to about 100 micromolar (μM) (e.g., from about 1 fM toabout 1 picomolar (pM), from about 1 pM to about 1 nanomolar (nM), fromabout 1 nM to about 1 micromolar (μM), or from about 1 μM to about 100μM). In one embodiment, the IL-33-binding agent can bind to an IL-33protein with a K_(D) less than or equal to 1 nanomolar (e.g., 0.9 nM,0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM,0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of theforegoing values). In another embodiment, the IL-33-binding agent canbind to IL-33 with a K_(D) less than or equal to 200 pM (e.g., 190 pM,175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or a rangedefined by any two of the foregoing values). Immunoglobulin affinity foran antigen or epitope of interest can be measured using anyart-recognized assay. Such methods include, for example, fluorescenceactivated cell sorting (FACS), separable beads (e.g., magnetic beads),surface plasmon resonance (SPR), solution phase competition (KINEXA™),antigen panning, and/or ELISA (see, e.g., Janeway et al. (eds.),Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 2001).

The IL-33-binding agent of the invention may be administered alone or incombination with other drugs (e.g., as an adjuvant). For example, theIL-33-binding agent can be administered in combination with other agentsfor the treatment or prevention of the diseases or disorders disclosedherein. In this respect, the IL-33-binding agent can be used incombination with at least one other anti-inflammatory agent including,for example, corticosteroids (e.g., prednisone and fluticasone) andnon-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin,ibuprofen, and naproxen).

In addition to therapeutic uses, the IL-33-binding agent describedherein can be used in diagnostic or research applications. In thisrespect, the IL-33-binding agent can be used in a method to diagnose adisease or disorder that is responsive to IL-33 inhibition orneutralization. In a similar manner, the IL-33-binding agent can be usedin an assay to monitor IL-33 protein levels in a subject being testedfor a disease or disorder that is responsive to IL-33 inhibition orneutralization. Research applications include, for example, methods thatutilize the IL-33-binding agent and a label to detect an IL-33 proteinin a sample, e.g., in a human body fluid or in a cell or tissue extract.The IL-33-binding agent can be used with or without modification, suchas covalent or non-covalent labeling with a detectable moiety. Forexample, the detectable moiety can be a radioisotope (e.g., ³H, ¹⁴C,³²P, ³⁵S, or ¹²⁵I), a fluorescent or chemiluminescent compound (e.g.,fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (e.g.,alkaline phosphatase, beta-galactosidase, or horseradish peroxidase), orprosthetic groups. Any method known in the art for separatelyconjugating an antigen-binding agent (e.g., an antibody) to a detectablemoiety may be employed in the context of the invention (see, e.g.,Hunter et al., Nature, 194: 495-496 (1962); David et al., Biochemistry,13: 1014-1021 (1974); Pain et al., J. Immunol. Meth., 40: 219-230(1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412 (1982)).

IL-33 protein levels can be measured using the inventive IL-33-bindingagent by any suitable method known in the art. Such methods include, forexample, radioimmunoassay (RIA), and FACS. Normal or standard expressionvalues of IL-33 can be established using any suitable technique, e.g.,by combining a sample comprising, or suspected of comprising, IL-33 witha IL-33-specific antibody under conditions suitable to form anantigen-antibody complex. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, and radioactive materials (see, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amountof IL-33 polypeptide expressed in a sample is then compared with astandard value.

The IL-33-binding agent can be provided in a kit, i.e., a packagedcombination of reagents in predetermined amounts with instructions forperforming a diagnostic assay. If the IL-33-binding agent is labeledwith an enzyme, the kit desirably includes substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides adetectable chromophore or fluorophore). In addition, other additives maybe included in the kit, such as stabilizers, buffers (e.g., a blockingbuffer or lysis buffer), and the like. The relative amounts of thevarious reagents can be varied to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders (typicallylyophilized), including excipients which on dissolution will provide areagent solution having the appropriate concentration.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example describes assays used to determine the functional activityof the inventive immunoglobulin heavy and light chain polypeptides.

IL-33-Mediated Release of IL5 from KU812 Cells.

KU812 cells, a human basophil-like CML cell line (ATCC No. CRL-2099)(see, e.g., Tare et al., Exp. Cell Res., 316(15): 2527-37 (2010);Lefrancais et al., Proc. Natl. Acad. Sci. USA, 109(5): 1673-1978(2012)), respond to IL-33 stimulation by secreting IL-5. KU812 cellswere suspended in RPM1+10% FBS culture medium, and 500,000 cells perwell were plated into 96-well flat bottom plates. A 30 μg/mL stock forantibody of interest was serially diluted to generate 8 concentrationsat half-log intervals. The diluted samples were added in rows to cellsand incubated at 37° C. for 30 minutes. IL-33-his6-bio (C-terminallylabeled with 6 His, and biotinylated with an average of 1 to 2 biotinsper molecule) (3 ng) was then added to each well, and plates wereincubated at 37° C. for 48 hours. Supernatants were then removed andheld at 4° C. until testing by ELISA. Supernatants were tested using anIL-5 DuoSet ELISA kit (R&D Systems, Minneapolis, Minn.) and evaluated ona SPECTRAMAX™ microplate reader (Molecular Devices, LLC, Sunnyvale,Calif.) using SOFTMAX PRO™ Microplate Data Acquisition & AnalysisSoftware (Molecular Devices, LLC, Sunnyvale, Calif.) to determine IL-5production.

IL-33-Mediated Expression of Luciferase in HEK293-ST2 Cells

An HEK/ST2-stable cell line was generated by first plating naïve HEKcells at 3×10³ cells/T75 flask in DMEM/10% FBS and incubating overnightat 37° C. The following day, cells were transfected by mixing 500 μLOptimem (Life Technologies, Carlsbad, Calif.)+24 μL HD FUGENE™ (Promega,Madison, Wis.) and allowed to incubate at room temperature for fiveminutes. DNA encoding ST2-Fc (4 μg) was added to the FUGENE™ mixture andallowed to incubate at room temperature for 25 minutes. The DNA/FUGENE™mixture was then distributed over the HEK cells and allowed to incubateovernight at 37° C., 5% CO₂. At 24 hours post transfection, cells weresplit and placed under hygromycin selection for a period of 3-4 weeksuntil stably selected.

IL-8 Luciferase Reporter Assay

4×10⁶ HEK293/ST2-Fc cells were seeded in a T-75 flask overnight at 37°C., 5% CO₂. The following morning, the DNA construct AB4111 whichencodes the human IL-8 promoter driving expression of a luciferasereporter gene, was transfected into the cells by mixing 500 μLOptimem+24 μL HD FUGENE™ and allowed to incubate at room temperature forfive minutes. The IL-8 promoter responds to the signal transductioncascade initiated by stimulation of the ST2-IL-1RAcP receptor complex byIL-33 occupancy. AB4111 (2 μs) was added to the FUGENE™ mixture andallowed to incubate at room temperature for 25 minutes. The DNA/FUGENE™mixture was then distributed over the HEK/ST2 cells and allowed toincubate for 8 hours. Cells were harvested with ACCUTASE™ and seededinto a 96-well, flat bottom plate, with 2.0×10⁴ cells per well in 0.1 mLDMEM/10% FBS. Plates were incubated for 15-18 hours at 37° C., 5% CO₂.The next morning, plates were gently inverted and tapped on paper towelsto remove media. 50 μL/well of fresh DMEM/10% FBS was added to eachwell. Cells were stimulated with pre-complexed IL-33/ST2-Fc or IL-33/Abfor 20 minutes at room temperature and then added to the cells andallowed to incubate for an additional 5 hours at 37° C. After 5 hours,luciferase activity was determined using the Steady Glo-Luciferase AssaySystem (Promega, Madison, Wis.) by adding luciferase reagent at 1:1vol/vol to each well. Wells were mixed and 150 μL/well was transferredto black-walled, clear-bottom plates and read on the ENVISTION™ PlateReader (PerkinElmer, Waltham, Mass.) using the Luminescence program(60-sec delay). Data was analyzed using a 4 parameter curve fit withGraphPad Prism 5 software (GraphPad, San Diego, Calif.).

Surface Plasmon Resonance (SPR) Methods

Binding kinetics and affinities of anti-IL33 antibodies were determinedby SPR on a BIACORE™ T200 instrument (GE Healthcare). Each of four flowcells on a Series S CM5 chip was immobilized with ˜10,000 RU anti-humanIgG (Fc). Antibodies (˜1 μg/mL) were captured for 60 seconds at a flowrate of 10 μL/min. Monomeric IL-33 was diluted in running buffer(HBS-EP+, pH 7.6) starting at approximately 100-fold higherconcentration than each antibody's KD. Each IL-33 concentration waspassed over all flow cells for 180 seconds at 30 μL/min, then allowed todissociate for 1800 seconds. Surfaces were regenerated with 3 M MgCl₂for 60 seconds. Association and dissociation kinetic constants (k_(on)and k_(off)) and steady-state affinity (KD) were derived from theresulting sensorgrams using BIACORE T200 Evaluation Software version1.0.

The results of the above assays with respect to several of theIL-33-binding agents described herein are shown in FIGS. 1A, 1B, 2A, and2B.

Example 2

This example describes experiments demonstrating the functional activityof an inventive IL-33-binding agent.

An immunoglobulin heavy chain polypeptide comprising the amino acidsequence of SEQ ID NO: 136 was paired with an immunoglobulin light chainpolypeptide comprising the amino acid sequence of SEQ ID NO: 171. Theresulting antibody was referred to as APE4909. The ability of APE4909 toinhibit IL-33-mediated release of IL-5 and IL-9 in primary humanbasophils was assessed as described below.

Leukocyte Reduction System (LRS) units processed from donor whole bloodwere obtained from the San Diego Blood Bank. Peripheral bloodmononuclear cells (PBMCs) were prepped by standard methods using Ficolldensity centrifugation separation. Approximately 10⁹ PBMCs weretypically obtained from an LRS unit. Basophils were isolated from PBMCsusing a human basophil isolation kit II (Miltenyi Bioteccat#130-092-662, San Diego, Calif.). The total yield of basophils wasapproximately 10⁶.

Basophils were diluted to a density of 2×10⁶/mL in RPMI 1640 mediumcontaining 10% fetal bovine serum, penicillin/streptomycin (P/S), and 25ng/mL of recombinant human IL-3 (R&D Systems, Minneapolis, Minn.). 100μL of diluted cells per well were plated in standard flat-bottom 96-welltissue culture plates for a final cell density of 2×10⁵ per well.Outside wells were filled with 200 μL PBS/well to minimize the effectsof non-uniform evaporation. Cells were cultured overnight in 5% CO₂ in a37° C. incubator.

The following day, APE4909 and a monomeric human ST2 protein(hST2—referred to as APE3906) were added at concentrations ranging from30 to 0 μg/mL serially diluted at half-log intervals in RPMI+10% FBS+P/Scontaining 50 ng/mL IL-33. Approximately 18 hours later, plates werecentrifuged at 300×g for 3 minutes. Supernatants were removed,transferred to a clean plate, and stored at −80° C. pending analysis.

IL-5 and/or IL-9 levels in the cell supernatants were assessed by ELISAusing a DUOSET™ ELISA kit (R&D Systems, Minneapolis, Minn.) followingthe manufacturer's suggested protocol. The APE4909 antibody inhibitedIL-33 mediated release of IL-5 and IL-9 in primary human basophils, asshown in FIGS. 3 and 4.

The results of this example demonstrate that the inventive IL-33-bindingagent can inhibit IL-33 activity.

Example 3

This example demonstrates the affinity of an inventive IL-33 bindingagent for IL-33.

The ability of the APE4909 antibody described in Example 2 to interactwith IL-33 was analyzed biophysically using a KINEXA™ 3200 biosensorplatform from Sapidyne Instruments (Boise, Id.). Binding experiments forhuman and cynomolgus IL-33 (cynoIL-33) were conducted as described belowand were run twice independently. Conditions for the first experimentare shown non-parenthetically, while conditions for the secondexperiment are provided parenthetically.

APE4909/Human IL-33

Solid phase was prepared using azlactone-coated beads coated using a 50μg/mL solution of histidine-tagged human IL-33. Binding experiments wereperformed in 1×PBS pH 7.4, 0.1% BSA. The APE4909 antibody at 10 pM (or20 pM) final concentration was incubated with IL-33 at 200 pM to 3.4 fM(or 400 pM to 6.7 fM) final concentrations for 3 (or 4) days at 4° C. 5mL (or 10 mL) of each mixture was applied to the beads coated with IL-33at a rate of 0.25 mL/min for 1200 seconds (or 2400 seconds). Freeantibody was detected with an ALEXAFLUOR™ 647-(Life Technologies,Carlsbad, Calif.) labeled donkey anti-human antibody. All data fit usingstandard KINEXA™ software.

APE4909/Cyno IL-33 (cIL-33)

Experiments were performed as described above for human IL-33, exceptthat the APE4909 antibody at 20 pM and 100 pM final concentration wasincubated with cIL-33 at 3 nM to 315 fM final concentrations for 24hours at 4° C. Each mixture was applied to the beads coated with cIL-33at a rate of 0.25 mL/min for 500 seconds (for 20 pM) or 2120 seconds(for 100 pM). Free antibody was detected with an ALEXAFLUOR™ 647-(LifeTechnologies, Carlsbad, Calif.) labeled donkey anti-human antibody. Thetwo curves were combined using N-curve analysis, and all data fit usingKINEXA™ software. To verify, the experiment was repeated at 200 pMAPE4909 antibody concentration using similarly prepared solid phase,buffers, and detection reagent. The APE4909 antibody was incubated withcIL-33 at 15 nM to 250 fM final concentrations for 24 hours at 4° C. andapplied to the beads coated with cIL-33 at a rate of 0.25 mL/min for 180seconds. This data fit using standard KINEXA™ software.

APE4909 affinities for human IL-33 and cynoIL-33 are shown FIG. 5 andFIG. 6, respectively. The results of this example demonstrate that theinventive IL-33-binding agent binds to both human IL-33 and non-humanprimate IL-33 with high affinity.

Example 4

This example demonstrates that certain inventive IL-33-binding agentscompete with the ST2 receptor for binding to human IL-33.

IL-33 binding was monitored using a BIACORE™ T200 system (GE Healthcare,Little Chalfont, Buckinghamshire, UK). Binding of IL-33 to various IL-33antibodies disclosed herein or the human ST2 receptor was addressed bycapturing an antibody and surveying the binding response of a fixedconcentration of IL-33 in combination with increasing amounts of ST2.Anti-human IgG (Fc-specific, ˜10,000 RU) was immobilized on a BIACORE™CM5 chip using EDC-activated amine coupling chemistry. The inventiveIL-33 antibodies or human ST2 fused to a human IgG1 Fc region (2.0μg/mL, 1 minute contact time at a flow rate of 10 μL/min) were thencaptured at 25° C. (˜300 RU) onto this surface. Next, an analytesolution (pre-incubated for greater than 30 minutes) containingmonomeric soluble human untagged IL-33 (1 nM) and untagged human ST2(10, 3.3, 1.1 or 0.37 nM) was flowed over captured ligands for 2 minutesat a rate of 30 μL/min, and dissociation was monitored for an additional2 minutes. The sensor chip surface was regenerated between each cycleusing 3M MgCl₂ (60 seconds at 30 μL/min).

A second set of experiments was performed as described above, but withthe following changes: (1) the sensor chip was immobilized withanti-mouse IgG (Fc-specific, ˜7500 RU); (2) human ST2 fused to murineIgG2aFc (1.0 μg/mL) was captured on the chip with a contact time of 4minutes; and (3) the pre-incubated analyte solution contained monomericsoluble human untagged IL-33 (10 nM) and either an inventive IL-33antibody or monomeric untagged human ST2 (100 nM, 25 nM, 6.3 nM or 1.6nM). Analyte solution continued to incubate for approximately 30 minuteswhile the machine performed startup cycles. Capture and analyte bindingwas performed in HBS-EP+ running buffer (10 mM HEPES, pH 7.6, 150 mMNaCl, 3 mM EDTA, 0.05% Surfactant P-20; Teknova).

ST2 binding to the same epitope of IL-33 as an inventive antibody wasassociated with a loss of binding response, as the pre-incubation of ST2with IL-33 would preclude access of the antibody to the epitope. ST2binding to a different epitope of IL-33 permits IL-33 to bind thecaptured inventive antibody, which was observed as an increase inbinding response since binding response is directly proportional to themass of the analyte/complex. The results of these experiments are setforth in Table 1.

TABLE 1 Inventive Antibody Response Conclusion APE00986 no response inthe does not bind IL-33 absence of ST2 APE02718 response decreases ST2binds same/overlapping as [ST2] increases epitope APE03833 responsedecreases ST2 binds same/overlapping as [ST2] increases epitope APE04269response decreases ST2 binds same/overlapping as [ST2] increases epitopeAPE05492 response increases ST2 binds different epitope as [ST2]increases

The results of this example demonstrate that certain inventiveIL-33-binding agents bind to an IL-33 epitope that is similar oridentical to the epitope bound by the ST2 receptor.

Example 5

This example demonstrates that an inventive IL-33-binding agent inhibitshuman IL-33-driven expansion of peripheral eosinophils.

IL-33 induces increased expression and release of IL-5 from CD4⁺ TH2cell populations, innate lymphoid type-2 cells (ILC2 cells), andbasophils. IL-5 is a cytokine that plays a key role in thedifferentiation, expansion, and survival of eosinophils, a population ofcells that is known to mediate certain aspects of atopic diseaseindications, such as asthma and rhinitis. In a preliminary study, humanIL-33 was injected intraperitoneally into wild-type Balb/c mice for sixconsecutive days at a dose of 5 μg per animal. Subsequent FACS analysisat the conclusion of this initial 6-day study indicated that humanIL-33-treated mice had elevated numbers of eosinophils in theirperipheral blood (as defined by high side-scatter analysis and CCR3Siglec-F and CD16/CD32 expression) as compared to vehicle (PBS)-treatedmice.

As a follow-up to the above study, wild-type Balb/c mice were againinjected with 5 μg human IL-33 daily for 6-days total (days 1-6), andthe anti-human IL-33 APE04909 antibody (described above) wasadministered on days −2 and +2 of the study at a dose of 10 mg/kg eachday. Similar groups of mice treated with human IL-33 at a dose of 5 μgdaily were administered either a control human IgG1 isotype mAb(designated APE00987) or human ST2-hFc fusion protein (designatedAPE027180), which represents a human IgG1 Fc-fusion dimeric version ofthe soluble IL-33 receptor (ST2). Both of these control proteins werealso administered at 10 mg/kg doses on days −2 and +2 of the study only,as described for the APE04909 antibody.

As shown in FIG. 7, the anti-IL-33 APE04909 antibody substantiallyinhibited human IL-33-driven eosinophil expansion in the peripheralblood compartment. In comparison, the human ST2-hFc protein failed tosignificantly reduce blood eosinophil numbers in human IL-33-treatedmice and did not show a reduced level of eosinophil numbers above thatdetected in mice treated with a control IgG mAb (APE00987).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An interleukin-33 (IL-33)-binding proteincomprising an immunoglobulin heavy chain comprising the CDR1, CDR2, andCDR3 of SEQ ID NO: 136, and an immunoglobulin light chain comprising theCDR1, CDR2, and CDR3 of SEQ ID NO:
 171. 2. The IL-33 binding protein ofclaim 1 comprising the immunoglobulin heavy chain variable region of SEQID NO: 136, and the immunoglobulin light chain variable region of SEQ IDNO:
 171. 3. The IL-33-binding protein of claim 1, wherein the IL-33binding protein is an antibody, an antibody conjugate, or anantigen-binding fragment thereof.
 4. The IL-33-binding protein of claim2, wherein the IL-33 binding protein is an antibody, an antibodyconjugate, or an antigen-binding fragment thereof.
 5. The IL-33-bindingprotein of claim 1, wherein the IL-33 binding protein is a F(ab′)2,Fab′, Fab, Fv, scFv, dsFv, dAb, or a single chain binding polypeptide.6. The IL-33-binding protein of claim 2, wherein the IL-33 bindingprotein is a F(ab′)2, Fab′, Fab, Fv, scFv, dsFv, dAb, or a single chainbinding polypeptide.
 7. The IL-33-binding protein of claim 1, whereinthe IL-33 binding protein is an IgG1 antibody.
 8. The IL-33-bindingprotein of claim 2, wherein the IL-33 binding protein is an IgG1antibody.
 9. A pharmaceutical composition comprising (a) theIL-33-binding protein of claim 1, and (b) a pharmaceutically acceptablecarrier.
 10. A pharmaceutical composition comprising (a) theIL-33-binding protein of claim 2, and (b) a pharmaceutically acceptablecarrier.
 11. A method of treating atopic dermatitis in a mammal, whichmethod comprises administering an effective amount of the IL-33 bindingprotein of claim 1 to the mammal.
 12. The method of claim 11, whereinthe IL-33 binding protein is an antibody, an antibody conjugate, or anantigen-binding fragment thereof.
 13. The method of claim 11, whereinthe IL-33 binding protein is a F(ab′)2, Fab′, Fab, Fv, scFv, dsFv, dAb,or a single chain binding polypeptide.
 14. The method of claim 11,wherein the half-life of the IL-33-binding protein in the mammal isbetween 30 minutes and 45 days.
 15. The method of claim 11, wherein theIL-33-binding protein binds to IL-33 with a K_(D) less than or equal to1 nanomolar.
 16. The method of claim 11, wherein the IL-33 bindingprotein is an IgG1 antibody.
 17. A method of treating atopic dermatitisin a mammal, which method comprises administering an effective amount ofthe IL-33 binding protein of claim 2 to the mammal.
 18. The method ofclaim 17, wherein the IL-33 binding protein is an antibody, an antibodyconjugate, or an antigen-binding fragment thereof.
 19. The method ofclaim 17, wherein the IL-33 binding protein is a F(ab′)2, Fab′, Fab, Fv,scFv, dsFv, dAb, or a single chain binding polypeptide.
 20. The methodof claim 17, wherein the half-life of the IL-33-binding protein in themammal is between 30 minutes and 45 days.
 21. The method of claim 17,wherein the IL-33-binding protein binds to IL-33 with a K_(D) less thanor equal to 1 nanomolar.
 22. The method of claim 17, wherein the IL-33binding protein is an IgG1 antibody.
 23. A nucleic acid encoding theimmunoglobulin heavy chain and/or immunoglobulin light chain of claim 1,optionally in a vector.
 24. A An isolated cell comprising the nucleicacid of claim
 23. 25. A nucleic acid encoding the immunoglobulin heavychain and/or immunoglobulin light chain of claim 2, optionally in avector.
 26. A An isolated cell comprising the nucleic acid of claim 25.27. A method of preparing the IL-33 binding protein of claim 1, themethod comprising expressing in a cell a nucleic acid encoding animmunoglobulin heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:136, and a nucleic acid encoding an immunoglobulin light chaincomprising CDR1, CDR2, and CDR3 of SEQ ID NO: 171, thereby preparing theIL-33 binding protein.
 28. A method of preparing the IL-33 bindingprotein of claim 2, the method comprising expressing in a cell a nucleicacid encoding the immunoglobulin heavy chain variable region of SEQ IDNO: 136, and the immunoglobulin light chain variable region of SEQ IDNO: 171, thereby preparing the IL-33 binding protein.